Reduction/oxidation (redox) is an important mechanism of post-translational modification controlling a wide variety of cellular functions. Reactive oxygen species (ROS) produced from various sources, such as mitochondrial leakage and NAD(P)H oxidases (Noxs), oxidize signaling molecules and transcription factors. Noxs are major enzymes responsible for production of superoxide (O2-), a component of ROS, at various subcellular localizations. O2- inactivates iron-sulfur cluster containing enzymes, thereby liberating free iron, which in turn generates the highly reactive hydroxyl radical. O2- produced in cells is rapidly converted to H2O2, while O2- also reacts with NO to form ONOO- , all of which act as ROS. Although the cellular function of plasma-membrane associated Noxs, such as Nox1 and p91phox (Nox2), has been extensively characterized, that of Nox4, an isoform of Nox expressed in the heart, is not well understood. Although oxidative stress in the heart is increased by aging and stress and during cardiac failure, the mechanism and localization of ROS production are not well understood. Our preliminary studies suggest that expression of Nox4 in the heart is upregulated by aging and pressure overload. However, the contribution of Nox4 to the increased oxidative stress and the progression of cardiac aging and heart failure remains to be elucidated. Furthermore, molecular mechanisms (targets) regulated by Nox4 and their contributions to growth and death responses in the heart are currently unknown. Interestingly, our preliminary results suggest that inhibition of Nox4 is protective during pressure overload but is detrimental during ischemia/reperfusion. The overall hypothesis in this proposal is that although physiological levels of Nox4 mediate cellular functions essential for cell survival under certain stress conditions, upregulation of Nox4 in aging and failing hearts elicits detrimental effects, such as increased production of ROS and subsequent mitochondrial dysfunction. In particular, our specific hypotheses are:1. Upregulation of Nox4 during aging and pressure overload increases oxidative stress, apoptosis and cardiac dysfunction, and is thereby detrimental. 2. Nox4 is a critical mediator of mitochondrial oxidative stress and mitochondrial dysfunction during heart failure. 3. The presence of a physiological level of Nox4 protects the heart from ischemia/reperfusion (I/R) injury. Nox4 plays an essential role in upregulating HIF-11 during acute ischemia, which in turn mediates stimulation of glycolysis, thereby preventing cardiac myocyte death during I/R. We will address these issues using newly generated mouse models of both gain and loss of function of Nox4, including Nox4 transgenic and KO mice, proteomic analyses and integrated physiology studies. Our results will elucidate the role of Nox4 in mediating both physiological and pathological functions in the heart during aging and under stresses. PUBLIC HEALTH RELEVANCE: Oxidation and reduction of proteins are important mechanisms regulating the function of proteins. During the past four years, we have been studying the function of thioredoxin 1, a small anti-oxidant, in the heart and demonstrated that thioredoxin1 is an essential regulator of protein oxidation/reduction in the heart and that thioredoxin1 negatively regulates cardiac hypertrophy by modifying cysteine residues of signaling molecules subjected to oxidation by hypertrophic stimuli. In this competing renewal, we will extend these observations and further elucidate the cellular functions modulated by oxidation and reduction of intracellular proteins in the heart. In particular, we will be focusing on NADPH oxidase 4 (Nox4), an enzyme producing superoxide, major reactive oxygen species in cells. Our preliminary results suggest that Nox4 is upregulated by aging and stress and mediates both physiological and pathological functions in the heart. Using newly generated genetically altered mouse models, together with the state of the art mouse physiology experiments, proteomic methods and NMR experiments, we will investigate the cardiac function of Nox4 and elucidate the molecular mechanisms by which Nox4 mediates both physiological and pathological functions in the heart. Our study will allow us to elucidate how the modification of cardiac proteins by oxidation and reduction affects physiological and pathological functions of the heart. The result obtained from this investigation may lead to development of a novel strategy to treat aging related heart diseases or congestive heart failure in patients by targeting oxidative modifications of specific cardiac proteins.